GSM8083941: B6_BM_NK_nanoCAGE_Lib1; Mus musculus; RNA-Seq
For all samples, total RNA was extracted using Quick-RNA Microprep Kit (Zymo Research, R1050). For spleen NK cells: 20ng of total RNA was used for each library. cDNA was generated from the RNA template using Template Switching RT Enzyme Mix (NEB, M0466) with hexamer priming. nanoCAGE-seq libraries were then prepared following the previously described protocol (Poulain, S. et al. Promoter Associated RNA, Methods and Protocols. Methods Mol Biology Clifton N J 1543, 57–109 (2017).). For each of the two B6 samples (Ly49D+ and Ly49D-), we also generated one additional library where we used 250ng total RNA as input, extracted mRNA, and further depleted truncated mRNA with Terminator™ 5'-Phosphate-Dependent Exonuclease (TER51020), which gave similar results For BM NK cells: A low-input protocol was used due to the low cell number (library 1: ~ 2000 cells; library 2: ~22,000 cells). Total RNA was extracted using Quick-RNA Microprep Kit (Zymo Research, R1050). cDNA was generated from the RNA template using NEBNext Single Cell/Low Input cDNA Synthesis & Amplification Module (NEB, E6421) with a Poly-T RT primer (20 µM): TAGTCGAACTGAAGGTCTCCGAACCGCTCTTCCGATCTTTTTTTTTTTTTTTTTTTTTVN and Template switching oligos (75 µM): TAGTCGAACTGAAGGTCTCCAGCA(N1:25252525)(N1)(N1)(N1)(N1)(N1)(N1)(N1)(N1)TATArGrGrG instead of the template switching oligos in the kit. (N1) represents one bp of the 8 bp UMI. 25252525 represents an equal mix of A/T/C/G. cDNA was then amplified using cDNA PCR primer forward and reverse (20 µM): TAGTCGAACTGAAGGTCTCCAGCA; TAGTCGAACTGAAGGTCTCCGAACC instead of the cDNA PCR primer in the kit. cDNA was quantified with Qubit and up to 0.5 ng of cDNA was applied for library preparation using Nextera XT library Prep Kit (Illumina, 15032350), following the previously described protocol (oulain, S. et al. Promoter Associated RNA, Methods and Protocols. Methods Mol Biology Clifton N J 1543, 57–109 (2017).). Libraries were size selected with double sided selection of 0.55x/0.95x Ampure beads and were sequenced on the Element AVITI machine to ~20 million reads (2 x 75 bp).